Chitosan derivatives for inactivation of endotoxins and surface protection of nanoparticles

ABSTRACT

The present disclosure provides a polymer comprising a derivative of chitosan, wherein the derivative is zwitterionic, as well as methods of using the polymer. In addition, the present disclosure provides a nanoparticle structure comprising a derivative of chitosan and a dendrimer, as well as methods of utilizing the nanoparticle structure.

PRIORITY CLAIM

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 61/539,557, filed on Sep. 27, 2011, theentire disclosure of which is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with government support under grant number NIHR21CA135130 awarded by the National Institutes of Health. The U.S.Government has certain rights in the invention.

BACKGROUND

Chitosan is a linear copolymer of D-glucosamine(2-amino-2-deoxy-D-glucose) and N-acetyl-D-glucosamine(2-acetamido-2-deoxy-D-glucose), obtained by partial (usually >80%)deacetylation of chitin, the main component of exoskeletons of insectsand crustaceans. Chitosan has a low oral toxicity (oral LD₅₀: >10,000mg/kg in mouse and >1500 mg/kg in rats) and has been used as a componentin various dietary supplements. In addition, chitosan is safe fortopical use and has been used as an ingredient of medical devices orcosmetics. Chitosan is considered to be a safe and biocompatiblematerial, and has been widely explored as a pharmaceutical excipient fora variety of applications such as wound healing, surgical adhesives,mucoadhesive oral drug/gene delivery, gene delivery, and tissueengineering.

Furthermore, chitosan is known to have a pKa of approximately 6.5.Therefore, chitosan is insoluble at a neutral pH but is positivelycharged and water-soluble at an acidic pH. Although the limitedsolubility of chitosan at a neutral pH is hypothesized to allow forformation of nanoparticle drug/gene delivery platforms, such limitedsolubility is disadvantageous for applications of a solution of chitosanat physiological conditions.

In addition, some studies suggest that chitosans exhibit harmfulbiological effects when administered parenterally. For example, chitosanhas been shown in some studies to cause a haemostatic effect andactivation of complement following administration to an animal Moreover,some studies suggest that chitosan induces pro-inflammatory cytokines orchemokines after administration. For example, intraperitoneal (IP)administration of chitosan has been shown to induce a large number ofmacrophages with hyperplasia in the mesenterium of mice and causessevere peritoneal adhesions in rabbits. In order for nanoparticles to becompatible with parenteral applications, the nanoparticles should notactivate immune cells in the bloodstream (monocytes, platelets,leukocytes, and dendritic cells) or in tissues (resident phagocytes)because such activation could cause premature removal of thenanoparticles from the body, and/or elicit inflammatory responses in thebody, following administration.

Therefore, there exists a need for chitosan derivatives that can besafely and effectively used as nanoparticles for parenteraladministration to animals. Moreover, new and effective methods ofutilizing a chitosan derivative, or compositions containing a chitosanderivative, are also very desirable. Accordingly, the present disclosureprovides chitosan derivatives and methods of using the chitosanderivatives that exhibit desirable properties and provide relatedadvantages for improvement in safety and efficacy after administration.

Additionally, chitosan derivatives may be advantageously utilized to aidin the delivery of other pharmaceutical compositions, such asdendrimers. Polyamidoamine (“PAMAM”) dendrimers have previously beenexplored as pharmaceutical compositions for the delivery of therapeuticor imaging agents. PAMAM dendrimers can have various functional groupson their surface, for example amines, carboxylates, andamidoethylethanolamines. In particular, amine-terminated PAMAMdendrimers may be useful for gene delivery to animals because of theircationic charge, which allows for complexation of nucleic acids and forcellular uptake of the dendrimers. Moreover, protonation of tertiaryamines in the interior of PAMAM dendrimers may facilitate endosomalescape via a “proton sponge” effect Amine-termini of PAMAM dendrimersmay also be useful for covalent conjugation of drugs via linkers cleavedby a condition unique to target tissues.

However, despite their ability to carry various agents, amine-terminatedPAMAM dendrimers are generally not useful for systemic applicationsbecause of the non-specific toxicity and high risk associated withuptake by the reticuloendothelial system (RES). In an attempt to reducethe charge-related toxicity and prevent opsonization of cationic PAMAMdendrimers, a portion of their amine termini may be modified withpolyethylene glycol (PEG), a hydrophilic linear polymer that masks thecationic charge. However, even after modification with polyethyleneglycol (i.e., “pegylation”), modified PAMAM dendrimers can bedisadvantageous due to the interference of PEG with the target cells.For example, PEG can interfere with important interactions between thecarrier and the target cell, causing cellular uptake of the dendrimersto potentially be reduced. Even further modification of the pegylatedPAMAM dendrimers with folate, transferrin, or RGD peptide (i.e., ligandsknown to enhance interactions with target cells) does not solve theproblems because the fraction of target cells that express correspondingcellular receptors is not always predictable, and the expression levelcan change during progression of the disease(s) to be treated.

Therefore, there exists a need for the modification of dendrimers (e.g.,PAMAM dendrimers) to provide for their safe and effective administrationto animals. Moreover, new and effective methods of utilizing suchmodified dendrimers are also very desirable. Accordingly, the presentdisclosure also provides a nanoparticle structure comprising aderivative of chitosan and a dendrimer, as well as methods of using thenanoparticle structure, that exhibit desirable properties and providerelated advantages for improvement in administering dendrimers toanimals.

The present disclosure provides a polymer comprising a derivative ofchitosan, wherein the derivative is zwitterionic, as well as methods ofusing the polymer. In addition, the present disclosure provides ananoparticle structure comprising a derivative of chitosan and adendrimer, as well as methods of utilizing the nanoparticle structure.

The chitosan derivative, the nanoparticle structure, and the methodsaccording to the present disclosure provide several advantages comparedto other compositions and methods in the art.

First, the chitosan derivative has a unique pH-dependent charge profile,with isoelectric points (pI) that are tunable among a pH range fromabout 4 to about 7. The chitosan derivative is zwitterionic and, thus,is soluble in water at pHs below and above the pI, according to thechange of its net charge. As a result, the zwitterionic chitosanderivative can be used for parenteral applications, specifically as acomponent of nanoparticulate drug delivery systems.

Second, the zwitterionic chitosan derivative demonstrates excellentcompatibility with blood components and is well tolerated following IPinjection. Compared to its precursors (e.g., low molecular weightchitosan), the zwitterionic chitosan derivative has a reduced potentialto cause hemolysis, complement activation, and pro-inflammatoryresponse.

Third, the zwitterionic chitosan derivative demonstrates a lowerincidence of causing tissue reactions and has a reduced propensity toinduce pro-inflammatory cytokine production from macrophages. Thezwitterionic chitosan derivative surprisingly suppressespro-inflammatory responses of activated macrophages.

Fourth, the zwitterionic chitosan derivative can advantageously be usedto decrease endotoxins in a composition or in a subject. Endotoxins area product of the cell wall of gram-negative bacteria and are a commoncause of toxic reactions due to their potent stimulation of themammalian immune system. It is believed that the zwitterionic chitosanderivative may be able to bind to lipopolysaccharides in order todecrease endotoxin levels. Thus, the zwitterionic chitosan derivativemay provide an efficient and cost-effective way of removing endotoxinfrom pharmaceutical products or may serve as a portable reagent forwater treatment, such as in war zones or underdeveloped countries.

Fifth, the zwitterionic chitosan derivative can be used to provide safedelivery of cationic polymer nanoparticles to cells by shielding them innormal conditions and activating them only upon exposure to commonfeatures of diseased tissues or organs. For example, themicroenvironment of a tumor could advantageously be used in this regard.Cancer cells distant from blood vessels are typically deprived of oxygenand undergo anaerobic glycolysis to generate excess lactic acid. As aresult, hypoxic tumors tend to develop a weakly acidic microenvironment(e.g., a pH of about 6.5 to about 7.2) compared to normal tissues.Accordingly, the zwitterionioc chitosan derivative of the presentdisclosure can be used to modify the surface of an amine-terminatedPAMAM dendrimer and shield its cationic surface of the dendrimer allowcellular entry in a pH-responsive manner In an acidic environment, thezwitterionic chitosan derivative can undergo charge reversal, thusallowing PAMAM to interact with tumor cells. For example, followingcharge reversal, the dendrimer could be effectively delivered to thecell, an agent carried by the dendrimer could be effectively deliveredto the cell, or a combination of both.

The following numbered embodiments are contemplated and arenon-limiting:

1. A nanoparticle structure comprising a derivative of chitosan and adendrimer.

2. The nanoparticle of clause 1, wherein the nanoparticle structure is acomplex of the derivative of chitosan and the dendrimer.

3. The nanoparticle of clause 2, wherein the complex is an electrostaticcomplex.

4. The nanoparticle of any one of clauses 1 to 3, wherein thenanoparticle structure has a ratio of derivative:dendrimer at about 1:1.

5. The nanoparticle of any one of clauses 1 to 3, wherein thenanoparticle structure has a ratio of derivative:dendrimer at about 2:1.

6. The nanoparticle of any one of clauses 1 to 3, wherein thenanoparticle structure has a ratio of derivative:dendrimer at about 3:1.

7. The nanoparticle of any one of clauses 1 to 3, wherein thenanoparticle structure has a ratio of derivative:dendrimer at about 4:1.

8. The nanoparticle of any one of clauses 1 to 7, wherein thenanoparticle structure has a critical association concentration betweenabout 2.0 μg/mL and about 3.0 μg/mL.

9. The nanoparticle of any one of clauses 1 to 8, wherein thenanoparticle structure has a critical association concentration of about2.5 μg/mL.

10. The nanoparticle of any one of clauses 1 to 8, wherein thenanoparticle structure has a critical association concentration of about2.7 μg/mL.

11. The nanoparticle of any one of clauses 1 to 10, wherein the size ofthe nanoparticle structure is between about 100 nm and about 500 nm

12. The nanoparticle of any one of clauses 1 to 10, wherein the size ofthe nanoparticle structure is between about 200 nm and about 400 nm

13. The nanoparticle of any one of clauses 1 to 12, wherein the size ofthe nanoparticle structure is about 200 nm

14. The nanoparticle of any one of clauses 1 to 12, wherein the size ofthe nanoparticle structure is about 250 nm

15. The nanoparticle of any one of clauses 1 to 12, wherein the size ofthe nanoparticle structure is about 300 nm

16. The nanoparticle of any one of clauses 1 to 12, wherein the size ofthe nanoparticle structure is about 350 nm

17. The nanoparticle of any one of clauses 1 to 12, wherein the size ofthe nanoparticle structure is about 400 nm

18. The nanoparticle of any one of clauses 1 to 17, wherein thederivative is zwitterionic.

19. The nanoparticle of any one of clauses 1 to 18, wherein thederivative has an isoelectric point (pI) between about 4 and about 7.

20. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 4.5.

21. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 5.0.

22. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 5.5.

23. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 6.0.

24. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 6.5.

25. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 6.8.

26. The nanoparticle of any one of clauses 1 to 19, wherein thederivative has a pI of about 7.0.

27. The nanoparticle of any one of clauses 1 to 26, wherein thederivative has an An/Am ratio between 0.3 to 0.7.

28. The nanoparticle of any one of clauses 1 to 27, wherein thederivative has an An/Am ratio of about 0.3.

29. The nanoparticle of any one of clauses 1 to 27, wherein thederivative has an An/Am ratio of about 0.4.

30. The nanoparticle of any one of clauses 1 to 27, wherein thederivative has an An/Am ratio of about 0.5.

31. The nanoparticle of any one of clauses 1 to 27, wherein thederivative has an An/Am ratio of about 0.6.

32. The nanoparticle of any one of clauses 1 to 27, wherein thederivative has an An/Am ratio of about 0.7.

33. The nanoparticle of any one of clauses 1 to 32, wherein thedendrimer is poly(amidoamine) (“PAMAM”).

34. The nanoparticle of clause 33, wherein the PAMAM dendrimer is anamine-terminated G5 PAMAM dendrimer.

35. A method of delivering a dendrimer to a cell, said method comprisingthe step of administering a nanoparticle structure comprising aderivative of chitosan and a dendrimer to the cell.

36. The method of clause 35, wherein the cell is a cancer cell.

37. The method of clause 35 or clause 36, wherein the nanoparticlestructure releases the dendrimer to the cell.

38. The method of clause 37, wherein the release occurs at an acidic pH.

39. The method of clause 38, wherein the acidic pH is caused by hypoxia.

40. The method of clause 38, wherein the acidic pH is caused by theWarburg effect.

41. The method of any one of clauses 36 to 40, wherein the delivery tothe cell is entry into the cell.

42. The method of clause 41, wherein the entry into the cell results inapoptosis of the cell.

43. The method of clause 42, wherein the apoptosis results from deliveryof the dendrimer to the cell.

44. The method of clause 42, wherein the apoptosis results from deliveryof an agent to the cell and wherein the agent is contained within thedendrimer or covalently conjugated to the dendrimer.

45. A method of delivering a dendrimer to a cell in a subject, saidmethod comprising the step of administering an effective amount of ananoparticle structure to the subject, wherein the nanoparticlestructure comprises a derivative of chitosan and a dendrimer.

46. The method of clause 45, wherein the cell is associated with a tumorin the subject.

47. The method of clause 46, wherein the tumor is a solid tumor.

48. The method of any one of clauses 45 to 47, wherein the nanoparticlestructure releases the dendrimer to the cell in the subject.

49. The method of clause 48, wherein the release occurs at an acidic pH.

50. The method of clause 49, wherein the acidic pH is caused by hypoxia.

51. The method of clause 50, wherein the acidic pH is caused by theWarburg effect.

52. The method of any one of clauses 45 to 51, wherein the delivery tothe cell is entry into the cell.

53. The method of clause 52, wherein the entry into the cell results inapoptosis of the cell.

54. The method of clause 53, wherein the apoptosis results from deliveryof the dendrimer to the cell.

55. The method of clause 54, wherein the apoptosis results from deliveryof an agent to the cell and wherein the agent is contained within thedendrimer or covalently conjugated to the dendrimer.

56. A method of delivering an agent to a subject, said method comprisingthe step of administering a nanoparticle structure to the subject,wherein the nanoparticle structure comprises a derivative of chitosan, adendrimer, and the agent.

57. The method of clause 56, wherein the agent is contained within thedendrimer or covalently conjugated to the dendrimer.

58. The method of clause 56 or clause 57, wherein the agent is deliveredto a cell in the subject.

59. The method of any one of clauses 56 to 58, wherein the agent is apharmaceutical compound.

60. The method of clause 59, wherein the pharmaceutical compound is ananticancer drug.

61. The method of any one of clauses 56 to 58, wherein the agent is animaging agent.

62. The method of any one of clauses 56 to 61, wherein the cell is acancer cell.

63. The method of any one of clauses 56 to 62, wherein the cell isassociated with a tumor in the subject.

64. The method of clause 63, wherein the tumor is a solid tumor.

65. The method of any one of clauses 56 to 64, wherein the nanoparticlestructure releases the dendrimer to the cell in the subject.

66. The method of clause 65, wherein the release occurs at an acidic pH.

67. The method of clause 66, wherein the acidic pH is caused by hypoxia.

68. A polymer comprising a derivative of chitosan, wherein thederivative is zwitterionic.

69. The polymer of clause 68, wherein the derivative has an isoelectricpoint (pI) between about 4 and about 7.

70. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 4.5.

71. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 5.0.

72. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 5.5.

73. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 6.0.

74. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 6.5.

75. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 6.8.

76. The polymer of clause 68 or clause 69, wherein the derivative has apI of about 7.0.

77. The polymer of any one of clauses 68 to 76, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

78. The polymer of any one of clauses 68 to 77, wherein the derivativehas an An/Am ratio of about 0.3.

79. The polymer of any one of clauses 68 to 77, wherein the derivativehas an An/Am ratio of about 0.4.

80. The polymer of any one of clauses 68 to 77, wherein the derivativehas an An/Am ratio of about 0.5.

81. The polymer of any one of clauses 68 to 77, wherein the derivativehas an An/Am ratio of about 0.6.

82. The polymer of any one of clauses 68 to 77, wherein the derivativehas an An/Am ratio of about 0.7.

83. A method of suppressing an inflammatory response in a subject, saidmethod comprising the step of administering an effective amount of apolymer to the subject, wherein the polymer comprises a zwitterionicderivative of chitosan.

84. The method of clause 83, wherein the inflammatory response isassociated with activated macrophages in the subject.

85. The method of clause 83 or clause 84, wherein the inflammatoryresponse is pro-inflammatory cytokine production.

86. The method of clause 85, wherein the cytokine production is IL-6production.

87. The method of clause 85, wherein the cytokine production is TNF-αproduction.

88. The method of clause 83 or clause 84, wherein the inflammatoryresponse is pro-inflammatory chemokine production.

89. The method of clause 88, wherein the chemokine production is MIP-2production.

90. The method of any one of clauses 83 to 89, wherein the derivativehas an isoelectric point (pI) between about 4 and about 7.

91. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 4.5.

92. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 5.0.

93. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 5.5.

94. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 6.0.

95. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 6.5.

96. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 6.8.

97. The method of any one of clauses 83 to 90, wherein the derivativehas a pI of about 7.0.

98. The method of any one of clauses 83 to 97, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

99. The method of any one of clauses 83 to 98, wherein the derivativehas an An/Am ratio of about 0.3.

100. The method of any one of clauses 83 to 98, wherein the derivativehas an An/Am ratio of about 0.4.

101. The method of any one of clauses 83 to 98, wherein the derivativehas an An/Am ratio of about 0.5.

102. The method of any one of clauses 83 to 98, wherein the derivativehas an An/Am ratio of about 0.6.

103. The method of any one of clauses 83 to 98, wherein the derivativehas an An/Am ratio of about 0.7.

104. A method of suppressing cytokine or chemokine production in asubject, said method comprising the step of administering an effectiveamount of a polymer to the subject, wherein the polymer comprises azwitterionic derivative of chitosan.

105. The method of clause 104, wherein the cytokine or chemokineproduction is associated with activated macrophages.

106. The method of clause 104 or clause 105, wherein the cytokine orchemokine production is induced by lipopolysaccharide (LPS).

107. The method of clause 106, wherein the polymer binds directly to theLPS.

108. The method of any one of clauses 104 to 107, wherein the cytokineis IL-6.

109. The method of any one of clauses 104 to 107, wherein the cytokineis TNF-α.

110. The method of any one of clauses 104 to 107, wherein the chemokineis MIP-2.

111. The method of any one of clauses 104 to 110, wherein the derivativehas an isoelectric point (pI) between about 4 and about 7.

112. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 4.5.

113. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 5.0.

114. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 5.5.

115. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 6.0.

116. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 6.5.

117. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 6.8.

118. The method of any one of clauses 104 to 111, wherein the derivativehas a pI of about 7.0.

119. The method of any one of clauses 104 to 118, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

120. The method of any one of clauses 104 to 119, wherein the derivativehas an An/Am ratio of about 0.3.

121. The method of any one of clauses 104 to 119, wherein the derivativehas an An/Am ratio of about 0.4.

122. The method of any one of clauses 104 to 119, wherein the derivativehas an An/Am ratio of about 0.5.

123. The method of any one of clauses 104 to 119, wherein the derivativehas an An/Am ratio of about 0.6.

124. The method of any one of clauses 104 to 119, wherein the derivativehas an An/Am ratio of about 0.7.

125. A method of binding a lipopolysaccharide, said method comprisingthe step of contacting the lipopolysaccharide with a polymer comprisinga zwitterionic derivative of chitosan.

126. The method of clause 125, wherein the derivative has an isoelectricpoint (pI) between about 4 and about 7.

127. The method of clause 125 or clause 126, wherein the derivative hasa pI of about 4.5.

128. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 5.0.

129. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 5.5.

130. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 6.0.

131. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 6.5.

132. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 6.8.

133. The method of any one of clauses 125 to 127, wherein the derivativehas a pI of about 7.0.

134. The method of any one of clauses 125 to 133, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

135. The method of any one of clauses 125 to 134, wherein the derivativehas an An/Am ratio of about 0.3.

136. The method of any one of clauses 125 to 134, wherein the derivativehas an An/Am ratio of about 0.4.

137. The method of any one of clauses 125 to 134, wherein the derivativehas an An/Am ratio of about 0.5.

138. The method of any one of clauses 125 to 134, wherein the derivativehas an An/Am ratio of about 0.6.

139. The method of any one of clauses 125 to 134, wherein the derivativehas an An/Am ratio of about 0.7.

140. A method of decreasing a bacterial toxin in a subject, said methodcomprising the step of administering an effective amount of a polymer tothe subject, wherein the polymer comprises a zwitterionic derivative ofchitosan.

141. The method of clause 140, wherein the bacterial toxin is anendotoxin.

142. The method of clause 140 or clause 141, wherein the derivative hasan isoelectric point (pI) between about 4 and about 7.

143. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 4.5.

144. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 5.0.

145. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 5.5.

146. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 6.0.

147. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 6.5.

148. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 6.8.

149. The method of any one of clauses 140 to 142, wherein the derivativehas a pI of about 7.0.

150. The method of any one of clauses 140 to 149, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

151. The method of any one of clauses 140 to 150, wherein the derivativehas an An/Am ratio of about 0.3.

152. The method of any one of clauses 140 to 150, wherein the derivativehas an An/Am ratio of about 0.4.

153. The method of any one of clauses 140 to 150, wherein the derivativehas an An/Am ratio of about 0.5.

154. The method of any one of clauses 140 to 150, wherein the derivativehas an An/Am ratio of about 0.6.

155. The method of any one of clauses 140 to 150, wherein the derivativehas an An/Am ratio of about 0.7.

156. A method of decreasing a bacterial toxin in a composition, saidmethod comprising the step of administering an effective amount of apolymer to the composition, wherein the polymer comprises a zwitterionicderivative of chitosan.

157. The method of clause 156, wherein the bacterial toxin is anendotoxin.

158. The method of clause 156 or clause 157, wherein the composition isa pharmaceutical composition.

159. The method of clause 156 or clause 157, wherein the composition iswater.

160. The method of any one of clauses 156 to 159, wherein the derivativehas an isoelectric point (pI) between about 4 and about 7.

161. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 4.5.

162. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 5.0.

163. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 5.5.

164. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 6.0.

165. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 6.5.

166. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 6.8.

167. The method of any one of clauses 156 to 160, wherein the derivativehas a pI of about 7.0.

168. The method of any one of clauses 156 to 167, wherein the derivativehas an An/Am ratio between 0.3 to 0.7.

169. The method of any one of clauses 156 to 171, wherein the derivativehas an An/Am ratio of about 0.3.

170. The method of any one of clauses 156 to 171, wherein the derivativehas an An/Am ratio of about 0.4.

171. The method of any one of clauses 156 to 171, wherein the derivativehas an An/Am ratio of about 0.5.

172. The method of any one of clauses 156 to 171, wherein the derivativehas an An/Am ratio of about 0.6.

173. The method of any one of clauses 156 to 171, wherein the derivativehas an An/Am ratio of about 0.7.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pH-dependent zeta-potential profiles of unmodified lowmolecular weight chitosan (LMCS) and zwitterionic chitosan (ZWC)derivatives prepared with different anhydride to amine (An/Am) ratios,chitosan glutamate (C-Gt), and glycol chitosan (Gly-C). Data areexpressed as averages with standard deviations of 3 repeatedmeasurements.

FIG. 2 shows chitosan precipitates (arrows) in the peritoneal cavity.Mice injected with chitosan glutamate intraperitoneally were examined 7days after injection. (A) Chitosan precipitates found between the liverand the stomach. (B) Chitosan precipitates stuck on the spleen (top) andthe liver (bottom). (C) Lobes of the liver were connected via chitosanresidue.

FIG. 3 shows hematoxylin and eosin staining of liver sections ofdifferent treatment groups. (A) PBS (100x); (B) Glutamate buffer (100×);(C) ZWC (100×); (D) Glycol chitosan (100×). (A-D) Normal capsularsurface (box). (E) Chitosan glutamate (100×): capsular surface of livermarkedly thickened with precipitates of chitosan, which are surroundedby chronic inflammation and mild fibrosis (box). (F) Chitosan glutamate(400×): precipitates of chitosan on the liver surface surrounded bymacrophages, fibroblasts, and neutrophils.

FIG. 4 shows the cytology of the peritoneal fluid from differenttreatment groups using hematoxylin and eosin staining. (A) PBS; (B)Glutamate buffer; (C) ZWC. (A-C) Peritoneal fluid composed of smallmacrophages (box) and lymphocytes (arrows). No chitosan precipitateswere identified. (D) Glycol chitosan: peritoneal fluid is composed oflarge macrophages (box) containing chitosan. (E) Chitosan glutamate:peritoneal fluid composed of large macrophages with intracellulareosinophilic chitosan. Extracellular chitosan (Ch) is surrounded bynumerous macrophages. All images are of 400× magnification.

FIG. 5 shows the viability of mouse peritoneal macrophages in thepresence of ZWC (An/Am ratio=0.7), chitosan glutamate (C-Gt) and glycolchitosan (Gly-C). Data are expressed as averages with standarddeviations of three repeated measurements. *: p<0.05; **: p<0.01 vs PBS.

FIG. 6 shows the of chitosan treatment (all in 2 mg/mL) on the levels ofproinflammatory cytokines released from (A) naïve mouse peritonealmacrophages and (B) LPS-challenged macrophages. Cytokine levels aredetermined by Milliplex Multi-Analyte Profiling cytokine/chemokinepanel. Media of the LPS-challenged macrophages were 10 times dilutedprior to analysis. Graphs on the right are displayed in narrow y-scales.ZWC (An/Am=0.7); C-Gt: chitosan glutamate; Gly-C: glycol chitosan. Dataare expressed as averages with standard deviations of three repeatedmeasurements. *: p<0.05; **: p<0.01; ***: p<0.001 vs PBS.

FIG. 7 shows the effect of chitosan treatment (all in 2 mg/mL) on theMIP-2 production from (A) naïve mouse peritoneal macrophages and (B)LPS-challenged macrophages. MIP-2 level is determined by ELISA. Media ofthe LPS-challenged macrophages were 10 times diluted prior to analysis.Data are expressed as averages with standard deviations of threerepeated measurements. **: p<0.01; ***: p<0.001.

FIG. 8 shows the effect of timed application of ZWC or LMCS (all in 2mg/mL) on MIP-2 production in the LPS-challenged macrophages. Mouseperitoneal macrophages were incubated with LPS for 0, 2, 4, 8, or 24hours, and the culture medium was sampled for determination of the MIP-2level (white bars). In another set, macrophages were incubated with LPSfor 0, 2, 4, or 8 hours with LPS and then treated with ZWC or LMCS, andthe media were sampled after 24 hours (grey or black bars). Data areexpressed as averages with standard deviations of three repeatedmeasurements.

FIG. 9 shows production from macrophages incubated with ZWC (2 mg/mL),LPS (1 μg/mL), a mixture of LPS (1 μg/mL) and ZWC (2 mg/mL) (LPS co-incw ZWC), or LPS pre-incubated with ZWC (equivalent to 1 μg/mL LPS underan assumption that all the LPS remained in the supernatant; LPS pre-incw ZWC). ELISA was performed on 10 times-diluted culture media. Data areexpressed as averages with standard deviations of three repeatedmeasurements. ***: p<0.001.

FIG. 10 shows the pH dependent zeta-potential profiles of ZWCderivative, PAMAM, and ZWC(PAMAM). ZWC_(0.3) and ZWC_(0.7) indicate ZWCderivatives prepared with an anhydride to amine ratio of 0.3 and 0.7,respectively. Each curve is a representative of at least 3 runs.

FIG. 11 shows absorbance (@ 660 nm) of ZWC(PAMAM) at different pHs.ZWC(PAMAM) was prepared with ZWC derivative (1 mg/mL) and PAMAM (0.5mg/mL). Data are expressed as averages with standard deviations of 3identically and independently prepared samples.

FIG. 12 shows the pH dependent dissociation of ZWC(PAMAM) upon pHdecrease from pH 7.4 to 3 by the addition of hydrochloric acid (left).The addition of NaCl that provided the same degree of ion increase anddilution effect without changing the pH did not induce significantdecrease in turbidity (right).

FIG. 13 shows stability of ZWC(PAMAM) incubated in different ionicstrengths for 48 hours, as measured with turbidity change. ZWC(PAMAM)prepared with (A) 1 mg/mL ZWC and 0.5 mg/mL PAMAM, and (B) 0.75 mg/mLZWC and 0.5 mg/mL PAMAM. Data are expressed as averages with standarddeviations of 3 identically and independently prepared samples. Therewas no difference between 0 and 48 hours in all samples (p>0.05).

FIG. 14 shows the determination of critical association concentration ofZWC(PAMAM). The result is representative of 3 independently andidentically prepared samples. The bottom plot shows a magnified view ofthe range indicated in the top plot.

FIG. 15 shows fluorescence profiles of (A) ZWC-552 (0.2 mg/mL) in thepresence of unlabeled PAMAM (0.05-0.2 mg/mL) at pH 7.4 (excited at 544nm; emission scanned from 550 to 650 nm) and (B) PAMAM-581 (0.2 mg/mL)in the presence of unlabeled ZWC (0.05-0.2 mg/mL) at pH 7.4 (excited at578 nm; emission scanned from 590 to 650 nm). Each plot isrepresentative of three replicates.

FIG. 16 shows the fluorescence profiles of (A) ZWC-552 (0.2 mg/mL) inthe presence of unlabeled PAMAM (0.05-0.2 mg/mL) at pH 9 (excited at 544nm; emission scanned from 560 to 650 nm), (B) ZWC-552 (0.2 mg/mL) in thepresence of unlabeled PAMAM (0.05-0.2 mg/mL) at pH 3 (excited at 544 nm;emission scanned from 560 to 650 nm), (C) PAMAM-581 (0.2 mg/mL) in thepresence of unlabeled ZWC (0.05-0.2 mg/mL) at pH 9 (excited at 578 nm;emission scanned from 600 to 650 nm), and (D) PAMAM-581 (0.2 mg/mL) inthe presence of unlabeled ZWC (0.05-0.2 mg/mL) at pH 3 (excited at 578nm; emission scanned from 600 to 650 nm). Each plot is representative ofthree replicates.

FIG. 17 shows transmission electron micrographs of PAMAM, ZWC, andZWC(PAMAM).

FIG. 18 shows hemolytic activity of ZWC, PAMAM, and ZWC(PAMAM). RBCswere incubated with the samples at concentrations shown in the table at37° C. and pH 7.4 for 1 hour. Pictures were taken after centrifugationof the tubes at 2000 rpm for 5 min The pellets are intact RBC, and thered supernatant or precipitate on the tube wall show hemoglobin releasedfrom the lysed RBC. Data are expressed as averages with standarddeviations of 3 identically and independently prepared samples. *:p<0.05 vs. PBS.

FIG. 19 shows the cell viability of ZWC, PAMAM, and ZWC(PAMAM) atvarious concentrations using MTT assay. Data are expressed as averageswith standard deviations of 3 repeated tests. #: p<0.005 vs. PAMAM (NoZWC) at each concentration. Numbers indicate the final concentrations ofZWC and/or PAMAM in culture medium

FIG. 20 shows the nuclei of cells treated with PAMAM or ZWC(PAMAM) at pH7.4 (top) and pH 6.4 (bottom): (A, D) cells only, (B, E) PAMAM (0.5mg/mL), and (C, F) ZWC(PAMAM) equivalent to PAMAM (0.5 mg/mL) and ZWC (1mg/mL).

DETAILED DESCRIPTION

Various embodiments of the invention are described herein as follows. Inone embodiment described herein, a nanoparticle structure is provided.The nanoparticle structure comprises a derivative of chitosan and adendrimer.

In another embodiment, a method of delivering a dendrimer to a cell isprovided. The method comprises the step of administering a nanoparticlestructure comprising a derivative of chitosan and a dendrimer to thecell.

In yet another embodiment, a method of delivering a dendrimer to a cellin a subject is provided. The method comprises the step of administeringan effective amount of a nanoparticle structure to the subject, whereinthe nanoparticle structure comprises a derivative of chitosan and adendrimer.

In a further embodiment, a method of delivering an agent to a subject isprovided. The method comprises the step of administering a nanoparticlestructure to the subject, wherein the nanoparticle structure comprises aderivative of chitosan, a dendrimer, and the agent.

In another embodiment, a polymer is provided. The polymer comprises aderivative of chitosan, wherein the derivative is zwitterionic.

In yet another embodiment, a method of suppressing an inflammatoryresponse in a subject is provided. The method comprises the step ofadministering an effective amount of a polymer to the subject, whereinthe polymer comprises a zwitterionic derivative of chitosan.

In a further embodiment, a method of suppressing cytokine production ina subject is provided. The method comprises the step of administering aneffective amount of a polymer to the subject, wherein the polymercomprises a zwitterionic derivative of chitosan.

In another embodiment, a method of binding a lipopolysaccharide isprovided. The method comprises the step of contacting thelipopolysaccharide with a polymer comprising a zwitterionic derivativeof chitosan.

In yet another embodiment, a method of decreasing a bacterial toxin in asubject is provided. The method comprises the step of administering aneffective amount of a polymer to the subject, wherein the polymercomprises a zwitterionic derivative of chitosan.

In a further embodiment, a method of decreasing a bacterial toxin in acomposition is provided. The method comprises the step of administeringan effective amount of a polymer to the composition, wherein the polymercomprises a zwitterionic derivative of chitosan.

In the various embodiments, the nanoparticle structure comprises aderivative of chitosan and a dendrimer. As used herein, the term“nanoparticle” refers to a particle having a size measured on thenanometer scale. As used herein, the “nanoparticle” refers to a particlehaving a structure with a size of less than about 1,000 nanometers. Asused herein, the term “chitosan” refers to a linear copolymer ofD-glucosamine (2-amino-2-deoxy-D-glucose) and N-acetyl-D-glucosamine(2-acetamido-2-deoxy-D-glucose), obtained by partial deacetylation ofchitin, the main component of exoskeletons of insects and crustaceans. A“derivative” of chitosan refers to refers to compound or portion of acompound that is derived from or is theoretically derivable fromchitosan. As used herein, the term “dendrimer” refers to a moleculebuilt up from a single starting molecule by sequential covalentreactions with a molecule having reactive sites to produce a branchedmolecule including terminal reactive groups. An example of the synthesisof a dendrimer is the synthesis of poly(amido-amine) (“PAMAM”)dendrimers including terminal amine groups, as described in Tomalia etal., Macromolecules, 19 2466 (1986); and U.S. Pat. No. 4,568,737 toTomalia et al., the disclosures of which are incorporated herein. Forexample dendrimers may be synthesized with 4, 8, 16, 32, 64, 128, 256,or more primary amine groups.

In some embodiments described herein, the nanoparticle structure is acomplex of the derivative of chitosan and the dendrimer. As used herein,the term “complex” refers to a molecular association, which can benon-covalent, between two molecular or atomic entities. In variousembodiments, the complex is an electrostatic complex.

In various embodiments described herein, the nanoparticle structure cancomprise various ratios of derivative to dendrimer(derivative:dendrimer). In one embodiment, the nanoparticle structurehas a ratio of derivative:dendrimer at about 1:1. In another embodiment,the nanoparticle structure has a ratio of derivative:dendrimer at about2:1. In yet another embodiment, the nanoparticle structure has a ratioof derivative:dendrimer at about 3:1. In another embodiment, thenanoparticle structure has a ratio of derivative:dendrimer at about 4:1.

In some embodiments described herein, the nanoparticle structure has aspecified critical association concentration (CAC). As used herein, theterm “critical association concentration” refers to the lowestconcentration at which components of the nanoparticle structure are ableto form a complex, for example an electrostatic complex. In oneembodiment, the nanoparticle structure has a critical associationconcentration of about 2.5 μg/mL. In another embodiment, thenanoparticle structure has a critical association concentration of about2.7 μg/mL.

In various embodiments described herein, the nanoparticle structures canhave a specified size. In one embodiment, the size of the nanoparticlestructure is between about 100 nm and about 500 nm In anotherembodiment, the size of the nanoparticle structure is between about 200nm and about 400 nm In yet another embodiment, the size of thenanoparticle structure is about 200 nm In one embodiment, the size ofthe nanoparticle structure is about 250 nm In another embodiment, thesize of the nanoparticle structure is about 300 nm. In yet anotherembodiment, the size of the nanoparticle structure is about 350 nm Inanother embodiment, the size of the nanoparticle structure is about 400nm

In some embodiments described herein, the derivative of chitosan iszwitterionic. As used herein, the term “zwitterionic” refers to amolecule that has both a negative and positive charges in the molecule,for example where the negative charge comes from the carboxyl group andthe positive charge comes from the amine group. For example, a“zwitterion” of chitosan may be produced by partial amidation ofchitosan with one or more compounds that provide anionic groups (e.g.,succinic anhydride). In some embodiments, the derivative has anisoelectric point (pI) between about 4 and about 7. In one embodiment,the derivative has a pI of about 4.5. In another embodiment, thederivative has a pI of about 5.0. In yet another embodiment, thederivative has a pI of about 5.5. In one embodiment, the derivative hasa pI of about 6.0. In another embodiment, the derivative has a pI ofabout 6.5. In another embodiment, the derivative has a pI of about 6.8.In yet another embodiment, the derivative has a pI of about 7.0.

In various embodiments described herein, the chitosan derivatives canhave a specified molar feed ratio of anhydride to amine (An/Am ratio).In one embodiment, the derivative has an An/Am ratio between 0.3 and0.7. In another embodiment, the derivative has an An/Am ratio of about0.3. In yet another embodiment, the derivative has an An/Am ratio ofabout 0.4. In another embodiment, the derivative has an An/Am ratio ofabout 0.5. In another embodiment, the derivative has an An/Am ratio ofabout 0.6. In yet another embodiment, the derivative has an An/Am ratioof about 0.7.

In various embodiments described herein, the dendrimer ispoly(amidoamine) (“PAMAM”). The core of a PAMAM dendrimer is a diamine(such as ethylenediamine), which is reacted with methyl acrylate, andthen another ethylenediamine to make the generation-0 (G-0) PAMAM.Successive reactions create higher generations. In some emdodiments, thePAMAM dendrimer is an amine-terminated generation 5 (G5) PAMAMdendrimer.

In one embodiment described herein, a method of delivering a dendrimerto a cell is provided. The method comprises the step of administering ananoparticle structure comprising a derivative of chitosan and adendrimer to the cell. The previously described embodiments of thenanoparticle structure are applicable to the method of delivering adendrimer to a cell described herein.

In some embodiments described herein, the step of “administering” may beadministered by any conventional route suitable for dendrimers,including, but not limited to, parenterally, e.g. injections including,but not limited to, subcutaneously or intravenously or any other form ofinjections or infusions. Formulations containing dendrimers can beadministered by a number of routes including, but not limited to oral,intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,topical, sublingual, intravascular, intramammary, or rectal means.Formulations containing dendrimers can also be administered vialiposomes. Such administration routes and appropriate formulations aregenerally known to those of skill in the art. Formulations containingdendrimers, alone or in combination with other suitable components, canalso be made into aerosol formulations (i.e., they can be “nebulized”)to be administered via inhalation. Aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations containing dendrimers suitable for parenteraladministration, such as, for example, by intraarticular (in the joints),intravenous, intramuscular, intradermal, intraperitoneal, andsubcutaneous routes, include aqueous and non-aqueous, isotonic sterileinjection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations containingdendrimers can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials. The formulations containingdendrimers can also be presented in syringes, such as prefilledsyringes.

In various embodiments described herein, the cell is a cancer cell. Inother embodiments, the nanoparticle structure releases the dendrimer tothe cell. As used herein, the term “release” refers to any mechanism bywhich the dendrimer can be delivered to the cell or cells of interest.In some embodiments, the release occurs at an acidic pH. The term“acidic pH” is well known in the art and includes any pH value below7.0. In some embodiments, the acidic pH is caused by hypoxia. The term“hypoxia” is well known in the art and refers to a lack of oxygen supplyto a particular area, for example to a cell or to a tissue. In otherembodiments, the acidic pH is caused by the Warburg effect. The Warburgeffect refers to a hypothesis that most cancer cells predominantlyproduce energy by a high rate of glycolysis followed by lactic acidfermentation in the cytosol, rather than by a comparatively low rate ofglycolysis followed by oxidation of pyruvate in mitochondria as in mostnormal cells.

In some embodiments described herein, the method of delivering adendrimer to a cell results in entry of the dendrimer into the cell. Invarious embodiments, the entry into the cell results in apoptosis of thecell. In one embodiment, the apoptosis results from delivery of thedendrimer to the cell. In another embodiment, the apoptosis results fromdelivery of an agent to the cell, wherein the agent is contained withinthe dendrimer or covalently conjugated to the dendrimer.

In one embodiment described herein, a method of delivering a dendrimerto a cell in a subject is provided. The method comprises the step ofadministering an effective amount of a nanoparticle structure to thesubject, wherein the nanoparticle structure comprises a derivative ofchitosan and a dendrimer. The previously described embodiments of thenanoparticle structure and of the method of delivering a dendrimer to acell are applicable to the method of delivering a dendrimer to a cell ina subject described herein.

In some embodiments described herein, the “subject” refers to a mammal.In other embodiments, the mammal is an animal. In yet other embodiments,the animal is a human.

As used herein, the term “effective amount” refers to an amount whichgives the desired benefit to a subject and includes both treatment andprophylactic administration. The amount will vary from one subject toanother and will depend upon a number of factors, including the overallphysical condition of the subject and the underlying cause of thecondition to be treated. The amount of dendrimer used for therapy givesan acceptable rate of change and maintains desired response at abeneficial level. A therapeutically effective amount of the presentcompositions may be readily ascertained by one of ordinary skill in theart using publicly available materials and procedures.

In various embodiments described herein, the cell is associated with atumor in the subject. In other embodiments, the tumor is a solid tumor.The terms “tumor” and “solid tumor” are well understood in the art ofoncology.

In one embodiment described herein, a method of delivering an agent to asubject is provided. The method comprises the step of administering ananoparticle structure to the subject, wherein the nanoparticlestructure comprises a derivative of chitosan, a dendrimer, and theagent. The previously described embodiments of the nanoparticlestructure, of the method of delivering a dendrimer to a cell, and of themethod of delivering a dendrimer to a cell in a subject are applicableto the method of delivering an agent to a subject described herein.

In some embodiments described herein, the agent is contained within thedendrimer or covalently conjugated to the dendrimer. In otherembodiments, the agent is delivered to a cell in the subject. In oneembodiment, the agent is a pharmaceutical compound.

In another embodiment, the pharmaceutical compound is an anticancerdrug. In yet another embodiment, the agent is an imaging agent. Thephrases “pharmaceutical compound,” “anticancer drug,” and “imagingagent” are well understood in the art. For example, a “pharmaceuticalcompound” refers to a substance used as a medication according to theFood, Drug and Cosmetic Act. The term “anticancer agent” includes anyagent that exhibits anti-tumor activity. Such agents include, withoutlimitation, chemotherapeutic agents (i.e., a chemical compound orcombination of compounds useful in the treatment of cancer), anticancerantibodies, agents that disrupt nucleic acid transcription and/ortranslation, such as antisense oligonucleotides, small interfering RNA(siRNA), and the like. The term “imaging agent” refers to a compoundthat is capable of localizing selectively at sites of diagnosticinterest in vivo such as at a particular organ, tissue or cell type.

In one embodiment described herein, a polymer is provided. The polymercomprises a derivative of chitosan, wherein the derivative iszwitterionic. The previously described embodiments of the nanoparticlestructure with respect to the derivative of chitosan are applicable tothe polymer described herein.

In one embodiment described herein, a method of suppressing aninflammatory response in a subject is provided. The method comprises thestep of administering an effective amount of a polymer to the subject,wherein the polymer comprises a zwitterionic derivative of chitosan. Thepreviously described embodiments of the polymer are applicable to themethod of suppressing an inflammatory response in a subject describedherein.

In various embodiments described herein, the inflammatory response isassociated with activated macrophages in the subject. The phrase“activated macrophage” is well known in the art, and includes cells thatsecrete inflammatory mediators and target and/or kill intracellularpathogens in the subject. In some embodiments, the inflammatory responseis pro-inflammatory cytokine production. Pro-inflammatory cytokines arewell known in the field of immunology and include, but are not limitedto, Interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12(IL-12), interferon-γ (IFN-γ), and tumor necrosis factor alpha (TNF-α).In one embodiment, the cytokine production is IL-6 production. Inanother embodiment, the cytokine production is TNF-α production.

In some embodiments, the inflammatory response is pro-inflammatorychemokine production. Pro-inflammatory chemokines are well known in theart of immunology. In one embodiment, the chemokine production isMacrophage inflammatory protein 2 (MIP-2) production.

In one embodiment described herein, a method of suppressing cytokine orchemokine production in a subject is provided. The method comprises thestep of administering an effective amount of a polymer to the subject,wherein the polymer comprises a zwitterionic derivative of chitosan. Thepreviously described embodiments of the polymer and of the method ofsuppressing an inflammatory response in a subject are applicable to themethod of suppressing cytokine or chemokine production in a subjectdescribed herein.

In various embodiments, the cytokine or chemokine production isassociated with activated macrophages. In some embodiments, the cytokineor chemokine production is induced by lipopolysaccharide (LPS). The term“lipopolysaccharide” is well known in the art, and refers to a moleculein which lipids and polysaccharides are linked, for example a componentof the cell wall of gram-negative bacteria. In some embodiments, thepolymer binds directly to the LPS.

In one embodiment described herein, a method of binding alipopolysaccharide is provided. The method comprises the step ofcontacting the lipopolysaccharide with a polymer comprising azwitterionic derivative of chitosan. The previously describedembodiments of the polymer, of the method of suppressing an inflammatoryresponse in a subject, and of the method of suppressing cytokine orchemokine production in a subject are applicable to the method ofbinding a lipopolysaccharide described herein.

In one embodiment described herein, a method of decreasing a bacterialtoxin in a subject is provided. The method comprises the step ofadministering an effective amount of a polymer to the subject, whereinthe polymer comprises a zwitterionic derivative of chitosan. Thepreviously described embodiments of the polymer, of the method ofsuppressing an inflammatory response in a subject, and of the method ofsuppressing cytokine or chemokine production in a subject are applicableto the method of decreasing a bacterial toxin in a subject describedherein.

In various embodiments, the bacterial toxin is an endotoxin. As usedherein, the term “endotoxin” refers to a toxin that is present inside abacterial cell (for example, a cell wall) and is released when the cellis broken down (e.g., dies).

In one embodiment described herein, a method of decreasing a bacterialtoxin in a composition is provided. The method comprises the step ofadministering an effective amount of a polymer to the composition,wherein the polymer comprises a zwitterionic derivative of chitosan. Thepreviously described embodiments of the polymer, of the method ofsuppressing an inflammatory response in a subject, of the method ofsuppressing cytokine or chemokine production in a subject, and ofdecreasing a bacterial toxin in a subject are applicable to the methodof decreasing a bacterial toxin in a composition described herein.

In some embodiments, the composition is a pharmaceutical composition. Inother embodiments, the composition is water.

While the invention has been illustrated and described in detail in theforegoing description, such an illustration and description is to beconsidered as exemplary and not restrictive in character, it beingunderstood that only the illustrative embodiments have been describedand that all changes and modifications that come within the spirit ofthe invention are desired to be protected. Those of ordinary skill inthe art may readily devise their own implementations that incorporateone or more of the features described herein, and thus fall within thespirit and scope of the present invention.

Example 1 Formation and Properties of Chitosan and Chitosan DerivativesSynthesis of Zwitterionic Chitosan Derivatives and Other Chitosans

In this example, chitosan derivatives were formed and analyzed. Inparticular, a zwitterionic chitosan (ZWC) derivative was synthesized.Briefly, low-molecular-weight chitosan (LMCS; MW: 15 kDa; degree ofdeacetylation: 87%; Polysciences) was first dissolved in 1% acetic acidto obtain an acetate salt form. LMCS acetate 200 mg was dissolved in 30mL of deionized water. Succinic anhydride was added as solid to the LMCSsolution under vigorous stirring varying the quantities according to thedesired molar feed ratio of anhydride to amine (An/Am ratio). The pH ofthe reaction mixture was maintained at 6-6.5 and subsequently increasedto 8-9 with 1 N NaHCO₃. After an overnight reaction at room temperatureunder stirring, the reaction mixture was dialyzed against water(molecular weight cutoff: 3500) maintaining the pH at 8-9 with 1 N NaOH.The purified ZWC was freeze-dried and stored at −20° C.

Chitosan Properties

All chitosan and chitosan derivatives showed pH-dependence in aqueoussolubilities and corresponding charge profiles (see FIG. 1). Solutionsof chitosan glutamate and LMCS (10 mg/mL) became turbid at pH above 6.5reaching the maximum turbidity at pH 8, where they had neutral charges.On the other hand, ZWC (10 mg/mL) formed clear solutions at both acidicand basic pHs, indicating aqueous solubility, except at the pI value.The pI value of ZWC decreased with the increase of the An/Am ratio (seeFIG. 1). Glycol chitosan was similar to chitosan glutamate and LMCS inthat it showed neutral charges around pH 8, but the solution (10 mg/mL)was not turbid. This suggests the aqueous solubility of glycol chitosan.

A summary of chitosan and various chitosan derivatives (collectivelyreferred to as “chitosans”) is provided in Table 1.

TABLE 1 Properties of Various Chitosans Degree of deacetylation AqueousMolecular (primary amine solubility Description Weight content) at pH7.4 Chitosan Glutamate 200 kDa 75-90%  Insoluble glutamate salt formGlycol chitosan 2-hydroxy-  82 kDa  83% Soluble ethylether derivative ofchitosan LMCS Parent of ZWC  15 kDa  87% Insoluble ZWC <29%^(b) ~15kDa >58% Soluble Derivative (An/Am = 0.3) ZWC >52   ~15 kDa <35% SolubleDerivative (An/Am = 0.7)

Example 2 In Vivo Properties of Chitosan and Chitosan Derivatives InVivo Biocompatibility and Gross Tissue Responses to IntraperitoneallyAdministered Chitosans

Chitosan glutamate, glycol chitosan, and ZWC were tested for tissueresponses following IP administration (800 mg/kg). Chitosan and buffercontrols (phosphate buffered saline (PBS), pH 7.4, or glutamate buffer,pH 5) were sterilized by aseptic filtration. Chitosan solutions (20mg/mL) were prepared by dissolving chitosan glutamate in water or glycolchitosan and ZWC in PBS. ICR mice (25 g) (Harlan, Indianapolis, Ind.)were anesthetized with subcutaneous injection of ketamine 50 mg/kg andxylazine 10 mg/kg. A 0.5 cm skin incision was made in the skin 0.5 cmabove the costal margin, and the peritoneum was nicked with a 24-gaugecatheter. One milliliter of 20 mg/mL chitosan solutions or controlbuffers were injected into the peritoneal cavity through the catheter,and the skin was closed with suture.

The animals were sacrificed after 7 days to evaluate the presence ofresidues, tissue adhesions, and visible signs of inflammation (nodules,increased vascularization) in the peritoneal cavity. Liver and spleenwere sampled for histology, and the peritoneal fluid was sampled on aslide for cytological analysis. After fixation in 10% formalin, thesectioned organ samples and peritoneal fluid cells were stained withhematoxylin and eosin (H&E).

Upon necropsy, the organs of animals treated with ZWC or glycol chitosanwere grossly normal. No material was found in the peritoneal cavity ofthe mouse injected with glycol chitosan or ZWC. On the other hand, whitechitosan precipitates were seen in all mice injected with chitosanglutamate due to the near-neutral pH of the peritoneal fluid (see FIG.2A). The white precipitates were usually present on the liver and spleen(see FIG. 2B). In 3 out of 4 cases, lobes of the liver were connectedvia the residual materials (see FIG. 2C).

Histological and Cytological Evaluation

Biomaterials delivered to peritoneal cavity often cause inflammatoryresponses followed by adhesion formation between in peritoneal tissuesand abdominal walls. Once entering systemic circulation, they can alsocause abnormalities in filtering organs. To estimate the destination andeffect of IP chitosan, peritoneal fluid and organs as well as abdominalwall were microscopically examined Incidence of lesions in peritonealtissues is summarized in Table 2.

TABLE 2 Incidence of lesions in tissues after intraperitoneal injectionof chitosans and buffers. Chitosan Glycol ZWC Derivative Glutamate PBSglutamate chitosan (An/Am = 0.7) buffer Liver,  0/2^(a) 4/4 0/4 0/5 0/3capsule inflammation Liver, 0/2 4/4 0/4 0/5 0/3 capsular chitosandeposits Spleen, 0/2 3/4 0/4 0/5 0/3 capsule inflammation Spleen, 0/23/4 0/4 0/5 0/3 capsular chitosan deposits Body wall, 0/2 3/4 1/4 0/50/3 inflammation Body wall, 0/2 0/4 0/4 0/5 0/3 chitosan depositsPeritoneal 0/2 4/4 3/3 0/5 0/4 fluid, inflammation Peritoneal 0/2 4/43/3 0/5 0/4 fluid, chitosan deposits ^(a)Incidence of occurrence: Numberof mice with lesion/total number of mice examined

In mice injected with PBS, glutamate buffer, and ZWC, no significantmicroscopic differences were seen in the liver (see FIGS. 3A, 3B, and3C), spleen, and abdominal wall. One mouse treated with glycol chitosanhad mild inflammation of the body wall, but liver (see FIG. 3D) andspleen were normal. Peritoneal tissues from other mice in this groupwere unremarkable. In contrast, mice treated with chitosan glutamate hadnoticeable chitosan precipitates on the liver, spleen and abdominalwall, which were surrounded by macrophages and neutrophils (see FIGS. 3Eand 3F). Capsular surface of the liver adjacent to precipitates ofchitosan was thickened and mildly fibrotic (see FIGS. 3E and 3F).

No abnormality was observed in peritoneal fluid of the animals injectedwith PBS, glutamate buffer, or ZWC (see FIGS. 4A, 4B, and 4C). However,chitosan precipitates were detected in peritoneal macrophages in micetreated with glycol chitosan (see FIG. 4D) or chitosan glutamate (seeFIG. 4E). Chitosan glutamate was also observed as extracellularresidues, surrounded by large activated macrophages (see FIG. 4E). Nochitosan precipitates were observed in those injected with ZWC (see FIG.4C).

Example 3 Chitosan Effect on Macrophage Proliferation

In an attempt to understand the difference in IP responses to chitosanglutamate, glycol chitosan, and ZWC, in vitro proliferation ofperitoneal macrophages was evaluated in the presence of the threechitosans. Peritoneal macrophages were chosen because they are prevalentin the peritoneal cavity and likely to be an important player ininflammatory responses to IP injected chitosans. Mouse peritonealmacrophages were maintained in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 5% fetal bovine serum and 5 mM HEPES. Cells wereseeded in 24 well plates at a density of 50,000 cells per well in 1 mLculture medium. After overnight incubation, chitosan solutions or 20mg/mL) were added to make a final concentration of the medium 0.2 or 2mg/mL. PBS and lipopolysaccharide (LPS) (1 μg/mL) were added in controlgroups. NUT assay was performed after 24 hours of incubation todetermine the effects of chitosans on macrophage proliferation.

For all chitosans, 0.2 mg/mL of chitosan treatment did not negativelyinfluence the macrophage proliferation (see FIG. 5). At 2 mg/mL, therewas a moderate reduction in macrophage proliferation with chitosanglutamate (p<0.01 vs. PBS). Glutamate buffer (pH 5) added in anequivalent volume showed a similar level of decrease in cellproliferation, indicating that this reduction might be partly due to theacidity of the medium. Neither glycol chitosan nor ZWC significantlyreduced the macrophage proliferation at 2 mg/mL.

Example 4 Cytokine Release From Peritoneal Macrophages Induced byChitosans

To investigate whether each chitosan had an intrinsic ability toactivate peritoneal macrophages, naïve (non-challenged) peritonealmacrophages were incubated with different chitosans (2 mg/mL), and themedium was analyzed to determine the concentrations of pro-inflammatorycytokines (IL-β, TNF-α, IL-6 and MIP-2). In this experiment, LMCS, theparent material for ZWC, was also tested.

Peritoneal macrophages were seeded in 24-well plates at a density of150,000 cells per well in 1 mL of medium. After overnight incubation,100 μL of the chitosan solution was added to each well to bring thefinal chitosan concentration in medium to 2 mg/mL. In control groups,100 μL of PBS or glutamate buffer (pH 5) was added in lieu of chitosansolutions. After 24 hour incubation, the culture media were centrifugedat 2000 rpm for 10 min to separate supernatants. The concentrations ofinterleukin (IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophageinflammatory protein (MIP)-2 in the supernatant were determined using aMilliplex Multi-Analyte Profiling (MAP) cytokine/chemokine panel(Millipore, Billerica, Mass.).

In another set of experiments, macrophages were first challenged byadding LPS to the media in the final concentration of 1 μg/mL shortlybefore the chitosans or buffer controls. For selected samples,enzyme-linked immunosorbent assay (ELISA) was performed to determine theMIP-2 levels using an MIP-2 ELISA kit (R&D systems, Minneapolis, Minn.).The detection range of MAP panel was 0-10,000 pg/mL for all analytes.For MIP-2 ELISA, standard curves were prepared in the range of 0-667pg/mL. In both assays, the supernatant collected from LPS-challengedmacrophages was always diluted 10 times prior to the analysis.

To investigate the time course of the ZWC effect on cytokine production,ZWC or LMCS was added in the final concentration of 2 mg/mL at 0, 2, 4,or 8 hours after the LPS addition. After incubating with chitosans for24 hours, the culture media were collected and diluted 10 times, and theMIP-2 levels were determined using ELISA. For comparison, another set ofmacrophages was challenged with LPS and incubated for 0, 2, 4, 8, or 24hours, and the media were sampled without any treatment or furtherincubation.

Briefly, 10 μg of LPS was mixed with 20 mg of ZWC in 1 mL of 0.9% NaCland incubated at room temperature for 1 hour. The ratio of LPS to ZWC(10 μg per 20 mg) was consistent with the ratio used in priorexperiments (1 μg per 2 mg). ZWC was then precipitated by decreasing thesolution pH to 4.8 with 0.1-1 M HCl and removed by 15-min centrifugationat 10,000 rpm. Assuming that the LPS was present in the supernatant, avolume equivalent to 1 μg of LPS was sampled and added to 1 mL ofperitoneal macrophage culture. After overnight incubation, MIP-2 levelsin the culture media were determined using ELISA.

To investigate whether each chitosan had an intrinsic ability toactivate peritoneal macrophages, naïve (non-challenged) peritonealmacrophages were incubated with different chitosans (2 mg/mL), and themedium was analyzed to determine the concentrations of pro-inflammatorycytokines (IL-1β, TNF-α, IL-6 and MIP-2). In this experiment, LMCS, theparent material for ZWC, was also tested. Naïve macrophages treated withPBS produced 37±7 pg/mL of MIP-2, 67±5 pg/mL of TNF-α, and 8±3 pg/mL ofIL-6, which were considered basal levels of cytokines. There was noadditional cytokine release in those treated with glutamate buffer,glycol chitosan, LMCS, and ZWC. There was no difference between LMCS andZWC-treated groups. On the other hand, chitosan glutamate treatmentresulted in significant increases in the levels of MIP-2 (p<0.001),TNF-α (p<0.05), and IL-6 (p<0.01), as compared with PBS-treatment (seeFIG. 6A).

To investigate how each chitosan influenced the cytokine production inactivated macrophages, the cells were first challenged with LPS, apotent inducer of cytokine release, prior to the addition of chitosans(2 mg/mL). LPS-challenged, then PBS-treated macrophages produced2341±564 pg/mL of MIP-2, 106±18 pg/mL of TNF-α, and 1346±535 pg/mL ofIL-6 (see FIG. 6B). Glutamate buffer caused increase in MIP-2production, whereas chitosan glutamate did not have any influence. LMCStreatment increased production of all three cytokines from theLPS-challenged macrophages. Interestingly, ZWC caused a marked decreasein the LPS-induced production of MIP-2 (p<0.01) and TNF-α (p<0.01) ascompared with PBS. Glycol chitosan also decreased the production ofMIP-2 as compared to PBS. Chitosan treatment did not cause any change inIL-113 levels in either naïve or LPS-challenged macrophages.

Example 5 MIP-2 Induction by Chitosans with a Varying Number of AmineGroups

The effects of chitosans on MIP-2 release from naïve or LPS-challengedmacrophages were monitored varying the amine content in the chitosan. Wecompared LMCS and ZWC with different An/Am ratios (0.3 or 0.7), all at 2mg/mL, with respect to the ability to induce macrophages to produceMIP-2, the most sensitive response in the prior experiment. From naïvemacrophages, LMCS induced a higher level of MIP-2 than PBS (p<0.01), butno significant change was observed after ZWC treatment (see FIG. 7A). InLPS-challenged macrophages, LMCS significantly increased the MIP-2 level(p<0.001). In contrast, the two ZWCs suppressed MIP-2 production fromthe LPS-challenged macrophages (p<0.001 for An/Am: 0.7, p<0.05 for An/Am0.3 vs. PBS) (see FIG. 7B). ZWC (An/Am: 0.7) decreased the LPS-inducedMIP-2 production to a greater extent than ZWC (An/Am: 0.3).

MIP-2 levels measured by ELISA were not identical to the valuesdetermined with the MAP panel, most likely due to the difference betweenthe two assay methods in the sensitive detection ranges. However,results of the two assays were consistent in that MIP-2 levels fromLPS-challenged macrophages were at least two orders of magnitude higherthan those of naïve macrophages and that the MIP-2 production from theLPS-challenged macrophages was significantly reduced by the ZWCtreatment.

Example 6 Onset of ZWC Derivative Effect on LPS-induced MIP-2 Production

To confirm the ability of ZWC to prevent LPS-induced cytokine productionand examine the onset of the action, macrophages were first challengedwith LPS for 0, 2, 4, or 8 hours. Subsequently, ZWC or LMCS were addedto the challenged macrophages, followed by incubation for additional 24hours.

In ZWC-treated macrophages, the MIP-2 levels in the culture media werecomparable to those sampled prior to ZWC treatment (see FIG. 8). Thisresult shows that cytokine production was completely blocked from thetime ZWC was added to the medium, and proliferating cells did notfurther produce cytokines. In contrast, LMCS-treated macrophagescontinued to produce MIP-2, resulting in the same level as those grownfor 24 hours without any other treatment after LPS challenge.

Example 7 LPS Inactivation by ZWC Derivative

To investigate how ZWC prevented the MIP-2 production from theLPS-challenged macrophages, LPS was incubated with ZWC for 1 hour beforeit was given to the macrophages. ZWC was removed by precipitation at pH4.8 (˜pI of ZWC) at the end of the 1-h incubation so that the directeffect of ZWC on the cells could be excluded.

FIG. 9 shows that the LPS-induced MIP-2 production was reduced when ZWCcoexisted in the culture, consistent with prior experiments. The LPSpre-treated with ZWC also lowered the MIP-2 production to a comparablelevel. This result suggests that the reduction in MIP-2 production wasdue to the inactivation of LPS by ZWC rather than a direct effect of ZWCon the LPS-challenged cells. A similar trend was observed with LPSpre-incubated at a higher ratio of LPS to ZWC (30 μg LPS per 20 mg ZWC).Further increase of LPS (40 μg LPS per 20 mg ZWC) resulted in asignificant production of MIP-2, indicating that there was an upperlimit of LPS that a fixed amount of ZWC could inactivate.

Example 8 Analysis of Endotoxins Using Chitosans

Biological activity of chitosan may be attributed to the positivecharges carried by the amine groups, which can electrostaticallyinteract with cell membranes or circulating plasma proteins and lead toplatelet adhesion/activation and thrombus formation. Due to the abilityto interact with serum proteins, chitosans activate macrophages andinduce cytokine production. Chitosan derivatives with reduced positivecharge densities cause much lower platelet adhesion and aggregation thanoriginal chitosan. Aqueous solubility of chitosan in physiological pH isalso expected to play a role in biological responses, because chitosanprecipitates can be subjected to phagocytic uptake and further stimulatemacrophages. Therefore, we hypothesized that the good hemocompatibilityof ZWC and the lack of pro-inflammatory effect might be related to thereduced amine contents of ZWC and/or the aqueous solubility at neutralpH.

The amount of endotoxin present in each chitosan was determined by thekinetic turbidometric Limulus Amebocyte Lysate (LAL) assay at Associatesof Cape Cod Inc. (East Falmouth, Mass.). Chitosan samples were initiallyprepared as 1 mg/mL (ZWC, LMCS) or 10 mg/mL (chitosan glutamate, glycolchitosan) solutions in LAL reagent water (LRW) and then serially dilutedfrom 1:20 to 1:8000 to find the minimum concentration that did notinterfere with analysis. E. coli O113:H10 was used as a control standardendotoxin and serially diluted from 0.32 to 0.002 EU/mL to construct acalibration curve. Positive product controls were prepared in parallelby fortifying the diluted samples with additional endotoxin equivalentto 0.008 EU/mL. LRW was tested as a negative control and found tocontain less than the lowest concentration of the calibration curve(0.002 EU/mL). Pyrotell®-T LAL lysate was reconstituted with Glucashieldbuffer, a β-glucan inhibiting buffer, and mixed with samples or controlsin a 1:1 ratio in a depyrogenated microplate. The absorbance of eachwell was monitored over time. The time required for the absorbance toincrease significantly over background was defined as the onset time.The correlation coefficient for the regression of log of onset time vs.log of endotoxin concentration was ≧0.98. All samples were tested induplicate. The results were reported as the amount of endotoxin presentin each chitosan (EU/g).

TABLE 3 Endotoxin levels in chitosans Sample Endotoxin concentration(EU/g) Chitosan glutamate 247 Glycol chitosan 311 LMCS 311 ZWCDerivative(An/Am = 0.3) 6,860 ZWC Derivative (An/Am = 0.7) 14,150

The levels were comparable among chitosan glutamate, glycol chitosan,and LMCS. However, endotoxin levels in ZWC derivative were one or twoorders of magnitude higher than those of other chitosans. ZWC derivativewith An/Am ratio 0.7 had highest endotoxin concentration. This resultsuggests a relatively high affinity of ZWC derivative to endotoxin.

Chitosans have been shown to induce production of pro-inflammatorycytokines or chemokines from macrophages. To examine if ZWC and glycolchitosan were intrinsically less bioactive than other chitosans, we thenmonitored the secretion of IL-1β, IL-6, TNF-α, and MIP-2 (murinefunctional homologue of IL-8) from peritoneal macrophages after treatingwith different chitosans. These cytokines or chemokines are responsiblefor both local and systemic inflammatory responses and have been used inevaluating the safety of other chitosan based formulations. Productionof MIP-2, IL-6, and TNF-α in naïve macrophages was increased bytreatment with chitosan glutamate but not with glycol chitosan, ZWC, orLMCS (see FIG. 6A). Chitosan glutamate is not particularly morecytotoxic than others; therefore, the difference is unlikely due to thechemotactic effect of dead cells.

The relatively high molecular weight of chitosan glutamate (200 kDa), ascompared to glycol chitosan (82 kDa), ZWC (15 kDa), and LMCS (15 kDa),may account for the relatively high pro-inflammatory effect of chitosanglutamate both in vivo and in vitro. The effect of the primary aminecontent on the intrinsic pro-inflammatory potential of chitosan is notreadily apparent from the MAP panel assay given the lack of differencebetween ZWC and LMCS (see FIG. 6A). ELISA detects a correlation betweenMIP-2 production and the amine content (LMCS >ZWC (An/Am=0.3)>ZWC(An/Am=0.7)), but the levels of MIP-2 are close to the basal level inall cases (see FIG. 7A). According to these results, ZWC and glycolchitosan have relatively low potential to cause inflammatory reactionsin the peritoneal cavity by themselves, and this property can beexplained by their aqueous solubility and relatively low molecularweights.

Additionally, chitosan could be administered to tissues with lesionsthat attract activated macrophages. Interestingly, Interestingly, onlyZWC suppressed the cytokine production from LPS-challenged macrophagessignificantly (see FIGS. 6B and 7B). Timed application of ZWC revealedthat MIP-2 production stopped as soon as ZWC was applied (see FIG. 8).

It is hypothesized that ZWC derivative may tightly bind to LPS andinactivate it, as evidenced by the fact that LPS pre-incubated with ZWCderivative lost the ability to induce MIP-2 (see FIG. 9). Moreover, ZWCderivatives show much higher endotoxin content than other chitosans,further suggesting that ZWC derivative has high affinity to LPS. The ZWCderivative-mediated inactivation of LPS appears to be potent andirreversible, given that ZWC derivative with such high endotoxin contentdid not activate naïve macrophages or induce inflammatory responses invivo. MIP-2 production from the LPS-challenged macrophages decreased inthe order of LMCS, ZWC derivative (An/Am=0.3), and ZWC derivative(An/Am=0.7) (see FIG. 7B), indicating that this ability may be relatedto the amine content (inversely proportional to the amidation degree,An/Am ratio) in chitosan.

Example 9 Formation and Properties of Chitosans, Chitosan Derivatives,and Nanoparticle Structures Synthesis of ZWC Derivative

In this example, ZWC derivative was synthesized according to thefollowing method. In short, chitosan acetate was suspended in deionized(DI) water, and succinic anhydride was added to the chitosan mixturewhile stirring. After an overnight reaction, the solution was dialyzed(molecular-weight cut off: 3500 Da) against water maintaining a pHbetween 10 and 11, and the purified ZWC derivative was lyophilized. ZWCderivative was re-suspended in deionized water (DI water) and reactedwith 30% H₂O₂ under vigorous stirring for 1 h at room temperature toproduce a lower molecular-weight ZWC derivative. The reaction wasquenched by the addition of methanol, and the resulting solution waspurified by dialysis. The purified product was lyophilized and stored at−20° C.

Preparation and Characterization of ZWC(PAMAM) Nanoparticle Structures

ZWC derivative solutions were prepared in phosphate buffers (pH 7.4)with ionic strengths, varying the concentration from 0.5 mg/mL to 2mg/mL. ZWC derivative (PAMAM) (“ZWC(PAMAM)”) nanoparticle structureswere created by mixing a small volume of PAMAM-methanol solution (40mg/mL) in the ZWC derivative solution achieving various ZWC derivativeto PAMAM ratios (1:1 to 4:1). The formation of ZWC(PAMAM) nanoparticlestructures was indicated by the development of turbidity, monitored at660 nm using a Beckman DU 650 UV-VIS Spectrophotometer (Brea, CA).Particle size of ZWC(PAMAM) nanoparticle structures was measured bydynamic light scattering using a Malvern Zetasizer Nano ZS90(Worchestershire, UK). Count rate (kilo counts per second), proportionalto the number of particles in solution, and polydispersity index, anindicator of the extent of particle aggregation, were also noted.Surface charges of ZWC(PAMAM) nanoparticle structures and eachcomponents were measured using a Malvern Zetasizer Nano ZS90 at pHranging from 3 to 9 in ˜0.3 increment. For this measurement, allcomponents and nanoparticle structures were prepared in 10 mM NaCl, andthe pH was adjusted using 0.1 N HCl or NaOH. pH-dependent chargeprofiles of PAMAM and ZWC Derivative Components

Zeta potentials of both ZWC derivative and PAMAM were measured at pHvalues ranging from 3 to 9. PAMAM (0.5 mg/mL) showed positive charges atall pH values (see FIG. 10). ZWC derivatives showed a negative charge ata relatively basic pH and a positive charge at an acidic pH. The pH atwhich the charge changed (transition pH) appears to correspond on theratio of succinic anhydride to chitosan. The transition pHs of ZWCderivative prepared with a An/Am ratio of 0.3 (ZWC_(0.3)) and 0.7(ZWC_(0.7)) were 6.6 and 4.3, respectively. Since ZWC_(0.7) was morelikely to form an electrostatic complex with PAMAM due to the strongernegative charge, ZWC_(0.7) was used in subsequent examples.

Formation of ZWC(PAMAM) Nanoparticle Structures

Upon introduction of ZWC derivative to PAMAM, the mixture immediatelybecame turbid, indicating the formation of nanoparticle structures. Thesuspension containing 1 mg/mL ZWC derivative and 0.5 mg/mL PAMAM showedan average particle size of 351.8 nm with a relatively narrow sizedistribution (PDI: 0.16) at a count rate of 1777.3 kilo counts per sound(kcps) (see Table 4). PAMAM as a 0.5 mg/mL colloidal solution in PBSshowed a particle size of 184.4 nm, but the count rate and PDI were 42.1kcps and 0.60, respectively. The low count rate and high PDI indicatedthat the observed particle size was due to the aggregation of PAMAM inwater, which has been reported in the literature. ZWC derivative (1mg/mL) showed a particle size of 535.2 nm with a similarly low countrate (62.5 kcps) and high PDI (0.76), suggesting that ZWC derivativealso aggregated when present alone in this concentration. The highparticle count rate of the PAMAM-ZWC mixture indicates that the twocomponents formed nanoparticle structures as complexes, which weredistinguished from each component, and the measured particle sizereflected that of the complexes rather than a simple sum of thecomponents.

TABLE 4 Particle size, polydispersity index, and derived count rate ofZWC(PAMAM) nanoparticle structures and the components. Particle sizePolydispersity Derived count Samples n (diameter, nm) index (PDI) rate(kcps) ZWC(PAMAM) 9 351.8 ± 21.1 0.16 ± 0.05 1777.3 ± 92.0 PAMAM 3 184.4± 25.0 0.60 ± 0.01  42.1 ± 46.3 ZWC derivative 3 535.2 ± 41.9 0.76 ±0.05  62.5 ± 3.8 * Samples prepared in phosphate-buffered saline (10 mMphosphate, pH 7.4). ** Each sample contained PAMAM 0.5 mg/mL and/or ZWC1 mg/mL.pH-Dependent Formation and Dissociation of ZWC(PAMAM) NanoparticleStructures

The ZWC(PAMAM) nanoparticle structures demonstrated a pH-dependentcharge profile, similar to that of ZWC, but with a transition pH shiftedto right from 4.3 to pH 6.8 (see FIG. 10). The increase in transition pHindicates partial neutralization of anionic charge of ZWC by PAMAM. Thenet charge of ZWC(PAMAM) nanoparticle structures at pH 7.4 wasapproximately −8 mV.

Turbidity of the suspension of nanoparticle structures decreased withthe decrease of pH (see FIG. 11). When pH was lowered to 3 by theaddition of HCl solution, the ZWC(PAMAM) suspension became completelyclear, similar to individual ZWC and PAMAM components, indicatingdissociation of the nanoparticle structures (see FIG. 12). However, theZWC(PAMAM) suspension diluted with NaCl solution to a comparable degreewhile keeping the pH at 7.4 did not show significant change inturbidity. This suggests that the dissociation of nanoparticlestructures observed at pH 3 was not due to dilution of the nanoparticlestructures or increase of ionic strength in the suspension. Given thatZWC assumes an increasingly positive charge as pH decreases, thedissociation of nanoparticle structures is most likely due toelectrostatic repulsion of protonated ZWC and PAMAM.

Example 10 Stability Evaluation of ZWC(PAMAM) Nanoparticle Structures

To study the effect of ionic strength on the formation and stability ofZWC(PAMAM) nanoparticle structures, the structures were suspended in pH7.4 phosphate buffers containing different concentrations of NaCl(10-300 mM) and incubated for 48 hours. ZWC(PAMAM) nanoparticlestructures were prepared by mixing 2 mg/mL ZWC solution inphosphate-buffered saline (PBS, 10 mM phosphate, pH 7.4) and 1 mg/mLPAMAM suspension in PBS, in equal volumes. The suspension was seriallydiluted by factors of 2 and 4 using PBS. Count rate of each suspensionwas obtained using dynamic light scattering (Malvern Zetasizer) with a 5mW He—Ne laser operated at 633 nm Count rates of ZWC and PAMAM solutionswere also measured at corresponding concentrations.

Turbidity of complex suspension decreased as the NaCl concentrationincreased, reaching a minimal value in 300 mM NaCl solution (see FIG.13). This result suggests that a large number of ions interfere with theformation of nanoparticle structures and, thus, confirms theelectrostatic nature of ZWC(PAMAM) nanoparticle structures. Thenanoparticle structures formed and incubated in 150 mM NaCl solutionmaintained a constant turbidity, particle size, and count rate over 48hours.

To examine the effect of dilution on stability of ZWC(PAMAM), thecritical association concentration (CAC) (i.e., the lowest concentrationat which ZWC and PAMAM formed electrostatic complexes) was determinedCAC was determined using dynamic light scattering as the concentrationabove which the intensity of scattered light (or particle count rate)showed a linear increase with concentration of the components. ZWC orPAMAM alone showed a minimal count rate, which did not change withconcentration, indicating the lack of particle formation (see FIG. 14).In contrast, ZWC(PAMAM) nanoparticle structures showed a linear increasein count rate with CAC concentrations corresponding to ZWC 1.8±0.3 μg/mLand PAMAM 0.9±0.2 μg/mL. Below this concentration, the count rateoverlapped with those of PAMAM alone, indicating dissociation ofZWC(PAMAM) nanoparticle structures.

Example 11 Elucidation of ZWC(PAMAM) Nanoparticle Structure withFluorescence Spectroscopy

The structure of ZWC(PAMAM) nanoparticles was elucidated by observingchanges in fluorescence emission profiles of (i) fluorescently labeledZWC (ZWC-552) in the presence of unlabeled PAMAM and (ii) fluorescentlylabeled PAMAM (PAMAM-581) in the presence of unlabeled ZWC. ZWC waslabeled with a fluorescent dye FPR-552 (λ_(abs): 551 nm; λ_(ex): 570 nm)per the manufacturer's protocol. Briefly, 1 mg of FPR-552 was dissolvedin a mixture of 50 μL dimethyl sulfoxide (DMSO) and 50 μL DI water, and1 mg of ZWC was dissolved in 100 μL of phosphate buffer (10 mM, pH 9).One microliter of the FPR-552 solution was incubated with 19 μL of theZWC solution overnight at room temperature in a dark environment, andexcessive dye was removed by dialysis. PAMAM was similarly labeled withan FPR-581 dye (λ_(abs): 578 nm; λ_(ex): 595 nm). The labeled ZWC andPAMAM were referred to as ZWC-552 and PAMAM-581, respectively.

Fluorescence spectra of ZWC-552, PAMAM-581, ZWC-552 combined withunlabeled PAMAM, and PAMAM-581 combined with unlabeled ZWC solutionswere obtained using a Molecular Devices SpectraMax M5 (Sunnyvale,Calif.). Samples containing ZWC-552 were excited at 544 nm with a cutoffof 550 nm, and their emission spectra were read from 550 to 650 nmSamples containing PAMAM-581 were excited at 578 nm with a 590 nmcutoff, and the emission spectra were read from 590 to 650 nm

At pH 7.4, a condition that allowed for attractive interaction betweenZWC derivative and PAMAM, ZWC-552 showed increasing fluorescenceintensity with increasing concentration of unlabeled PAMAM (see FIG.15A). In contrast, PAMAM-581 incubated with unlabeled ZWC derivativeshowed decreasing fluorescence intensity with increasing concentrationof unlabeled ZWC derivative (see FIG. 15B).

The increasing fluorescence intensity of ZWC-552 with increasing PAMAMmay be explained by de-quenching of ZWC-552, which was present asaggregates by themselves but dissociated upon complexation withPAMAM-552. This explanation is supported by the lack of suchfluorescence change at pH 9 (see FIG. 16A), where ZWC derivative had astronger anionic charge and was less likely to self-associate than ZWCderivative at pH 7.4. To the contrary, fluorescence intensity ofPAMAM-581 decreased as the amount of ZWC derivative increased. Thisresult suggests that emission of PAMAM-581 fluorescence might have beenblocked due to coverage by ZWC derivative. A similar trend was seen atpH 9 (see FIG. 16C), where anionic ZWC derivative and cationic PAMAM-581were supposed to form an electrostatic complex. These distinct changesin fluorescence intensity of ZWC-552 and PAMAM-581 in the presence ofunlabeled counterparts were not seen at pH 3 (see FIGS. 16B and 16D),where both ZWC derivative and PAMAM were charged positively and thus didnot form ionic complexes. These results indicate that ZWC(PAMAM)complexes are formed by electrostatic interactions between the twocomponents, in which PAMAM is covered by ZWC derivative.

Example 12 Transmission Electron Microscopy (TEM) Evaluation ofZWC(PAMAM) Nanoparticle Structures

ZWC (0.5, 1 and 2 mg/mL), PAMAM (0.5 mg/mL), and ZWC(PAMAM) nanoparticlestructures (specified concentrations) were prepared in DI water at pH7.4. Samples were mounted on a 400-mesh Cu grid with formvar and carbonsupporting film (not glow-discharged) and stained with 2% uranyl acetate(UA) solution. Excess stain was removed with filter paper, and the gridwas dried prior to imaging. Samples were imaged using a Philips CM-100TEM (FEI Company, Hillsboro, Oreg.) operated at 100 kV, spot size 3, 200um condenser aperture, and 70 μm objective aperture. Images werecaptured using a SIA L3-C 2 megapixel CCD camera (Scientific Instrumentsand Application, Duluth, Ga.) at original microscope magnificationsranging from 25,000× to 180,000×.

ZWC(PAMAM) nanoparticle structures and each individual component werevisualized with TEM after UA staining (see FIG. 17). PAMAM and ZWC wereoppositely stained by UA, which is likely due to the differentialaffinity of UA for each component. UA breaks down into different acetateion species, which react with both anionic and cationic groups, but witha much greater affinity for anionic groups such as phosphoryl andcarboxyl groups. Therefore, ZWC (which is anionic at pH 7.4) waspositively stained, whereas cationic PAMAM (which lacks phosphoryl andcarboxyl groups) appeared lighter (negatively stained). In the absenceof ZWC, PAMAM was observed as round white particles with a size <10 nmin diameter. In the sample prepared with ZWC and PAMAM at a 2:1 or 4:1ratio, dark ZWC appeared around light PAMAM. In contrast, at a 1:1ratio, numerous PAMAM appeared separately from ZWC, indicatingincomplete ZWC coverage of PAMAM.

Example 13 Hemolytic Activity and Cytotoxicity of ZWC(PAMAM)Nanoparticle Structures

To investigate the effect of ZWC coating, the hemolytic activity ofZWC(PAMAM) nanoparticle structures was compared with that of PAMAM.First, blood was collected from Spague-Dawley rats via the dorsal aorta.Then red blood cells (RBC) were isolated from blood and washed using 210mM NaCl solution until the supernatant became free of red color.Purified RBC pellets were incubated with 900 μL of ZWC, PAMAM, orZWC(PAMAM) nanoparticle structures in PBS at various concentrations for1 h at 37° C. DI water (positive control) caused complete lysis in thiscondition. PBS was used as a negative control. Samples were centrifugedat 2000 rpm for 5 minutes following incubation. 980 μL of supernatantwas removed, and the remaining RBC pellet was dissolved in 980 μL of DIwater. Absorbance of the RBC solution was measured at 541 nm Data wereexpressed as normalized to the PBS-treated RBC.

As shown in FIG. 18, ZWC alone (at concentrations of 0.5-2 mg/mL) had nohemolytic effect on RBC. However, PAMAM at 0.5 mg/mL showed significanthemolysis, and ZWC(PAMAM) nanoparticle structures containing 0.5 mg/mLZWC and 0.5 mg/mL PAMAM (ZWC:PAMAM=1:1) showed RBC lysis to a similarextent. On the other hand, ZWC(PAMAM) nanoparticle structures formed athigher ZWC:PAMAM ratios (between 2:1 to 4:1) exhibited no hemolysis.This result suggests that ZWC coating prevented direct interactionbetween PAMAM and RBC.

In addition, the cytotoxicity of ZWC, PAMAM, and ZWC(PAMAM) nanoparticlestructures was evaluated using NIH 3T3 mouse fibroblast cells (ATCC,Rockville, Md.) via an MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.Fibroblasts were cultured in DMEM high glucose medium supplemented with10% bovine calf serum (ATCC, Rockville, MD), 100 U/mL penicillin and 100μg/mL streptomycin. For the MTT assay, cells were seeded in 96-wellplates at a density of 10,000 cells per well. After overnightincubation, culture medium was replaced with various concentrations ofZWC (0.5, 1, 1.5, 2 mg/mL), PAMAM (0.05, 0.1, 0.5 mg/mL), or ZWC(PAMAM)nanoparticle structures (formed with combinations of ZWC and PAMAMconcentrations) suspended in PBS containing 10% calf serum.

After 4 hours of incubation with the samples, the media was replacedwith 100 μL of fresh medium and 15 μL of 5 mg/mL MTT, and the incubationwas continued for 3.5 hours. The stop/solubilization solution was thenadded to dissolve the formed formazan. To avoid the interference due toturbidity of ZWC(PAMAM) nanoparticle structures, plates were centrifugedfor 30 minutes at 4000 rpm, and clear supernatant was collected prior toreading. Cell viability was estimated by reading the absorbance of thesolubilized formazan in the supernatant at 562 nm The obtainedabsorbance was normalized to the absorbance of cells grown in completemedium without any treatment.

The protective effect of ZWC was confirmed by the MTT assay (see FIG.19). ZWC had minimal cytotoxic effects on fibroblasts at allconcentrations. In contrast, cell viabilities decreased to 36%, 18%, and4% of non-treated control cells at 0.05, 0.1, and 0.5 mg/mL PAMAM,respectively. The cytotoxicity of PAMAM decreased with the addition ofZWC at the concentrations of 0.5-2 mg/mL for each level of PAMAM. At thelower PAMAM concentrations, cell viability was improved upon addition ofZWC 0.5 mg/mL, from 36% (0.05 mg/mL PAMAM) and 18% (0.1 mg/mL PAMAM) to80% and 76%, respectively, which were comparable to the viability at ZWC0.5 mg/mL alone. The viability did not increase beyond this level athigher concentrations of ZWC, indicating that 0.5 mg/mL ZWC wassufficient for shielding between 0.05-0.1 mg/mL PAMAM. For 0.5 mg/mLPAMAM, cell viability gradually increased in a dose-dependent mannerwith the increase of ZWC concentration, reaching 60% with 2 mg/mL ZWC.This result indicates that ZWC coating can protect blood cells from thetoxic effect of PAMAM.

Example 14 Confocal Microscopy Evaluation of ZWC(PAMAM) NanoparticleStructures

To test the pH-dependent removal of ZWC derivative coating from aZWC(PAMAM) nanoparticle structure, cell responses to the nanoparticlestructure were observed at different pHs (7.4 and 6.4) using confocalmicroscopy. SKOV-3 ovarian carcinoma cells (ATCC, Rockville, MD) werecultured in RPMI-1640 medium supplemented with 10% fetal bovine serum(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells wereplated in 35 mm diameter glass bottom dishes at a density of 800,000 perdish. After overnight incubation, the medium was replaced with asuspension of PAMAM or ZWC(PAMAM) nanoparticle structures. Here, thePAMAM sample was prepared in PBS, and the ZWC(PAMAM) nanoparticlestructures were prepared in PBS by mixing ZWC with PAMAM at a 2:1 ratio.The suspensions were supplemented with 10% FBS, and their pH wasadjusted to 7.4 or 6.4 before adding to the cells. The finalconcentration of each component in the suspensions was 1 mg/mL ZWCderivative and/or 0.5 mg/mL PAMAM. After incubation with the treatmentsfor 1 hour at 37° C., cells were washed twice in PBS (pH 7.4) orpH-adjusted PBS (pH 6.4) and imaged in each buffer containing 1 μL ofDRAQS nuclear stain (Axxora, San Diego, Calif.). DRAQS was excited with633 nm laser and images of cell nuclei were obtained using an OlympusFV1000 confocal microscope using a 60× objective.

At both pHs, SKOV-3 cells treated with 0.5 mg/mL PAMAM showed punctatesignals around the nuclei (see FIGS. 20B and 20E), representing nucleifragmentation, which may be attributable to the pro-apoptotic effect ofPAMAM. The cells treated with ZWC(PAMAM) at pH 7.4 (see FIG. 20C) werecomparable to buffer-treated ones (see FIGS. 20A and 20D) with no signsof abnormality. In contrast, the punctate nuclear signals were seen inthe cells treated with ZWC(PAMAM) at pH 6.4 (see FIG. 20F), similar tothose treated with PAMAM alone (see FIGS. 20B and 20E).

1. A nanoparticle structure comprising a derivative of chitosan and adendrimer.
 2. The nanoparticle of claim 1, wherein the nanoparticlestructure is an electrostatic complex of the derivative of chitosan andthe dendrimer.
 3. The nanoparticle of claim 2, wherein the nanoparticlestructure has a critical association concentration of about 2.7 μg/mL.4. The nanoparticle of claim 1, wherein the derivative is zwitterionic.5. The nanoparticle of claim 1, wherein the derivative has anisoelectric point (pI) between about 4 and about
 7. 6. The nanoparticleof claim 1, wherein the dendrimer is poly(amidoamine) (“PAMAM”).
 7. Amethod of delivering a dendrimer to a cell in a subject, said methodcomprising the step of administering an effective amount of ananoparticle structure to the subject, wherein the nanoparticlestructure comprises a derivative of chitosan and a dendrimer.
 8. Themethod of claim 7, wherein the cell is associated with a tumor in thesubject.
 9. The method of claim 8, wherein the nanoparticle structurereleases the dendrimer to the cell in the subject and wherein therelease occurs at an acidic pH.
 10. The method of claim 7, wherein thedelivery to the cell results in apoptosis of the cell.
 11. The method ofclaim 10, wherein the apoptosis results from delivery of the dendrimerto the cell.
 12. The method of claim 10, wherein the apoptosis resultsfrom delivery of an agent to the cell and wherein the agent is containedwithin the dendrimer or covalently conjugated to the dendrimer.
 13. Amethod of suppressing cytokine or chemokine production in a subject,said method comprising the step of administering an effective amount ofa polymer to the subject, wherein the polymer comprises a zwitterionicderivative of chitosan.
 14. The method of claim 13, wherein the cytokineor chemokine production is associated with activated macrophages. 15.The method of claim 13, wherein the cytokine or chemokine production isinduced by lipopolysaccharide (LPS).
 16. The method of claim 13, whereinthe polymer binds directly to the LPS.
 17. The method of claim 13,wherein the cytokine is IL-6.
 18. The method of claim 13, wherein thecytokine is TNF-α.
 19. The method of claim 13, wherein the chemokine isMIP-2.
 20. The method of claim 13, wherein the derivative has anisoelectric point (pI) between about 4 and about 7.